A variety of methods and routes of administration have been developed to deliver pharmaceuticals, small molecular drugs and biologically active compounds such as peptides, hormones, proteins, and enzymes, to their site of action. Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, and intralymphatic injections that use a needle or catheter. The blood circulatory system provides systemic spread of the pharmaceutical. Polyethylene glycol and other hydrophilic polymers have been used to provide protection and to increase the circulatory time of the pharmaceutical in the blood stream by preventing interaction with blood components and preventing opsonization, phagocytosis and uptake by the reticuloendothelial system. For example, the enzyme adenosine deaminase has been covalently modified with polyethylene glycol to increase the circulatory time and persistence of this enzyme in the treatment of patients with adenosine deaminase deficiency. The controlled release of pharmaceuticals after their administration is under intensive development.
In the rational design of synthetic delivery vehicles for biologically-active compounds and macromolecules, the problem of providing for endosomal escape can be a critical barrier to efficient delivery to cytoplasmic or nuclear sites of action. Various methods have been employed in attempts overcome this barrier including: liposomes, which hypothetically fuse with cell endosomal membranes; viruses; which may either fuse with or rupture endosomal membranes; and polymer-based or other non-viral systems; which must destabilize or rupture endosomal membranes. Currently, none of these systems affects efficient escape of co-endocytosed material from internal membrane enclosed compartments. Understanding of endosomal release and ability to synthetically enhance it remain a largely unsolved process.
Both viral and synthetic processes for accomplishing endosomal release often rely upon acidification of the endosome and/or lysosome to trigger either membrane fusion or disruption. For viruses, the reduced pH of the endocytic compartment triggers a conformational change that induces endosomal escape. The pH gradient between cytoplasm and endosome also causes monoamines such as chloroquine to concentrate within endosomes thus destroying the pH gradient. The pH-sensitive amines present on polyamines such as PEI may also play a role in endosomal release although it is unclear what role protonation plays. For some liposome-based systems, pH-sensitive groups have been incorporated into lipids that enable the lipid to undergo phase transformations upon protonation. Finally, peptides and synthetic polymers containing protonatable groups have been modeled after viral sequences to become more amphipathic and membrane active in acidic environments. By limiting membrane activity to acidic vesicles, effects on the plasma membrane, and thereby cellular toxicity, are theoretically attenuated.
Unfortunately, the use of protonation to effect membrane disruption is beset with a theoretical conundrum: Endosome disruption destroys the pH-gradient (i.e. membrane integrity is essential to the maintenance of a pH gradient). With loss of a pH gradient, activity of the pH-dependent endosomalytic agent is reversed. Thus, delivery is potentially limited by the endosomal membrane resealing before macromolecules are able to diffuse out. The use of linkages that are labile within an endosomal or lysosomal milieu has previously been used in liposomes or for coupling drugs with carriers (Blattler et al. 1985). Specifically, citraconic anhydride has been used to reversibly modify the primary amine of DOPE (dioleoylphosphatidylethanolamine) (Reddy and Low 2000). However, the kinetics of reversal of this reagent are too slow to be effective in cells. Furthermore, this compound was found to be ineffective in inhibiting membrane activity of endosomalytic agents to which it was attached. In order to address the problems associated with currently available endosomalytic agents, we have developed a process of rapid irreversible activation that relies upon chemical bond cleavage to unmask a biological agent's activity.
In addition to endocytosis-dependent delivery systems, cell penetrating compounds that do not rely on endocytosis for delivery of compounds to the interior of a cell have been recently described. Delivery by these carriers involves attachment of the compound to a highly cationic, arginine-rich peptide (Lindgren et al. 2000). One such import peptide, GRKKRRQRRR (SEQ ID 1), is derived from the TAT protein of the HIV virus. This peptide, when attached to a variety of molecules facilitates their intracellular delivery both in vitro and in vivo (Schwarze et al. 1999; Lee and Pardridge 2001). Other import molecules include peptides from VP22, a herpes simplex virus protein, and the ANT protein, (RQIKIWFQNRRMKWKK, SEQ ID 2) derived from the homeo-domain of the Drosophila transcription factor antennapedia. These peptides contain no obvious homology other than the high content of cationic residues, especially arginine. In fact, peptides and peptide analogs composed solely of arginine residues have been shown to have import properties (Futaki 2002). For all of these peptides, import does not appear to occur via endocytosis since import occurs readily at the endocytosis impermissible temperature of 4° C. However, delivery of molecules by conjugation to these cationic import peptides is not selective. This lack of specificity results in organ distribution of TAT-modified streptavidin that is similar to the distribution of unconjugated streptavidin (Lee and Pardridge 2001). The compounds and processes we have developed for use with membrane active compounds also functions in controlling the activity of the cell penetrating compounds.